Methods for absolute quantification of low-abundance polypeptides using mass spectrometry

ABSTRACT

The present invention provides methods for improved label-free absolute quantification of relatively low abundant polypeptides by liquid chromatography/mass spectrometry analysis of peptide products obtained from simple or complex polypeptide mixtures. The methods for absolute quantification include MS signals from a set of qualified ions of peptide products of a relatively high abundant polypeptide to improve quantification of a relatively low abundant polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/996,031, filed on Jun. 1, 2018, which claims priority benefit to U.S.Provisional Application No. 62/514,587, filed on Jun. 2, 2017, thedisclosure of each of which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present invention provides methods for absolute quantification oflow-abundance polypeptides by liquid chromatography/mass spectrometry(LC/MS) analysis of peptide products obtained from simple or complexpolypeptide mixtures.

BACKGROUND OF THE INVENTION

A diverse array of mass spectrometry (MS)-based techniques forpolypeptide quantification are known in the art. For example,polypeptides may be quantified using metabolic-based techniques (e.g.,stable isotope labeling using amino acids in cell culture (SILAC)),peptide standard-based techniques (e.g., selected reaction monitoring(SRM) and multiple reaction monitoring (MRM)), and label-basedtechniques (e.g., Tandem Mass Tags (TMT)). These methods have welldocumented drawbacks, such as limited sample sources for SILAC,extensive development and cost of SRM and MRM, and the additional sampleprocessing and yield of relative abundances of label-based techniques.

MS-based label-free quantification techniques were developed to simplifyMS-based polypeptide quantification methods and to circumvent some ofthe above-mentioned limitations. However, current label-freequantification techniques may suffer from low accuracy and highvariability, and most label-free techniques may only provide a relativequantification ratio between two or more samples (e.g., spectralcounting).

One approach for MS-based label-free absolute quantification of proteinsinvolves using a protein standard to create a single point calibrationmeasurement that is applied to subsequent mass spectrometry analyses forthe absolute quantification of other proteins. J. C. Silva et al., MolCell Proteomics, 5, 144-56, 2006; U.S. Pat. No. 8,271,207. However, useof a single point calibration and differences created by separateenzymatic digestions of the sample and the protein standard may be majorsources of quantification variability for this method.

Thus, there is a need in the art for improved MS-based label-freeabsolute quantification techniques that are sensitive, accurate, andprecise, and can be applied to a diverse array of polypeptide samples ina high-throughput manner.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for label-free absolutequantification of low-abundance polypeptides in a sample comprisinganother polypeptide of relative high abundance. For example, theinvention provides methods for quantifying low-abundance host cellproteins in a culture system, or downstream product thereof, comprisinga host cell capable of producing a recombinant polypeptide, such as atherapeutic polypeptide. The methods use the concentration and peptideproduct MS signals of a high abundance polypeptide and peptide productMS signals of a low-abundance polypeptide to calculate the absolutequantity of the low-abundance polypeptide.

In one aspect, the present invention provides methods for absolutequantification of a first polypeptide in a sample comprising a pluralityof polypeptides comprising the first polypeptide and a secondpolypeptide, wherein the first polypeptide is at least 10-fold lower inabundance than the second polypeptide, the method comprising: (a)analyzing peptide products of the plurality of polypeptides at aplurality of sample loading quantities using a liquidchromatography/mass spectrometry (LC/MS) technique to obtain MS signalsof ions of the peptide products of the plurality of polypeptides at eachof the plurality of sample loading quantities, wherein the plurality ofsample loading quantities comprises a first sample loading quantity anda second sample loading quantity, and wherein the first sample loadingquantity is greater than the second sample loading quantity; (b)calculating the average or sum of an MS signal for: (i) a top set of nnumber of qualified ions of peptide products of the first polypeptidewith the highest MS signals at the first sample loading quantity (A);(ii) a top set of n number of qualified ions of peptide products of thesecond polypeptide with the highest MS signals at the second sampleloading quantity (B); (iii) a middle set of m number of qualified ionsof peptide products of the second polypeptide at the first sampleloading quantity (C); and (iv) the middle set of qualified ions ofpeptide products of the second polypeptide at the second sample loadingquantity (D), wherein A, B, C, and D are all calculated using theaverage or all calculated using the sum of the MS signal; and (c)determining an absolute quantity of the first polypeptide in the firstsample loading quantity based on the following formula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof.

In another aspect, the present invention provides methods for absolutequantification of a first polypeptide in a sample comprising a pluralityof polypeptides comprising the first polypeptide and a secondpolypeptide, wherein the first polypeptide is at least 10-fold lower inabundance than the second polypeptide, the method comprising: (a)obtaining MS signals of ions of peptide products of the plurality ofpolypeptides, wherein said MS signals of ions of the peptide productsare obtained by analyzing the peptide products of the plurality ofpolypeptides using a liquid chromatography/mass spectrometry (LC/MS)technique, wherein MS signals of the peptide products are obtained foreach of a plurality of sample loading quantities comprising a firstsample loading quantity and a second sample loading quantity, andwherein the first sample loading quantity is greater than the secondsample loading quantity; (b) calculating the average or sum of an MSsignal for: (i) a top set of n number of qualified ions of peptideproducts of the first polypeptide with the highest MS signals at thefirst sample loading quantity (A); (ii) a top set of n number ofqualified ions of peptide products of the second polypeptide with thehighest MS signals at the second sample loading quantity (B); (iii) amiddle set of m number of qualified ions of peptide products of thesecond polypeptide at the first sample loading quantity (C); and (iv)the middle set of qualified ions of peptide products of the secondpolypeptide at the second sample loading quantity (D), wherein A, B, C,and D are all calculated using the average or all calculated using thesum of the MS signal; and (c) determining an absolute quantity of thefirst polypeptide in the first sample loading quantity based on thefollowing formula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof.

In some embodiments, the average of the MS signal is used fordetermining the absolute quantity of the first polypeptide.

In some embodiments, the sum of the MS signal is used for determiningthe absolute quantity of the first polypeptide.

In some embodiments, the middle set of qualified ions of peptideproducts of the second polypeptide is selected based on quantificationerror of the qualified ions of peptide products of the secondpolypeptide from the plurality of sample loading quantities, or thefirst sample loading quantity and/or the second sample loading quantity.

In another aspect, the present invention provides methods for selectinga set of qualified ions of peptide products for absolute quantificationof a first polypeptide in a sample comprising a plurality ofpolypeptides comprising the first polypeptide and a second polypeptide,wherein the first polypeptide is at least 10-fold lower in abundancethan the second polypeptide, the method comprising: (a) analyzing thepeptide products of the plurality of polypeptides at a plurality ofsample loading quantities using a liquid chromatography/massspectrometry (LC/MS) technique to obtain MS signals of ions of thepeptide products of the plurality of polypeptides at each of theplurality of sample loading quantities, wherein the plurality of sampleloading quantities comprises a first sample loading quantity and asecond sample loading quantity, and wherein the first sample loadingquantity is greater than the second sample loading quantity; and (b)selecting a middle set of m number of qualified ions of peptide productsof the second polypeptide, wherein the middle set of qualified ions ofpeptide products of the second polypeptide is selected based onquantification error of the qualified ions of peptide products of thesecond polypeptide from the plurality of sample loading quantities, orthe first sample loading quantity and/or the second sample loadingquantity. In some embodiments, the methods further comprise selecting atop set of n number of qualified ions of peptide products of the secondpolypeptide with the highest MS signals at the second sample loadingquantity.

In some embodiments, each of the top set of qualified ions of peptideproducts is different than each of the middle set of qualified ions.

In some embodiments, the MS signal is ionization intensity or peakheight or peak area or peak volume.

In some embodiments, the methods further comprise obtaining the sample.

In some embodiments, the sample has been purified or enriched. In someembodiments, the methods further comprise processing the plurality ofpolypeptides in the sample to produce the peptide products. In someembodiments, processing the sample comprises one or more of thefollowing: (a) centrifuging the sample to isolate the plurality ofpolypeptides; (b) purifying the plurality of polypeptides in the sample;(c) removing from the sample components incompatible with subsequentprocessing and the mass spectrometry analysis; (d) digesting theplurality of polypeptides to produce the peptide products; and (e)purifying the peptide products.

In some embodiments, the LC/MS technique comprises separating thepeptide products via a liquid chromatography technique.

In some embodiments, the LC/MS technique comprises processing theobtained MS signals of the peptide products.

In some embodiments, the LC/MS technique further comprises one or morethe following: (a) identifying the peptide products by amino acidsequence; (b) identifying the first polypeptide by a protein identifier;and (c) identifying one or more of the plurality of polypeptides by aprotein identifier.

In some embodiments, the methods further comprise determining theabsolute quantity of the second polypeptide.

In some embodiments, the first polypeptide is a host cell protein or abiomarker. In some embodiments, the first polypeptide is at least100-fold lower in abundance than the second polypeptide.

In some embodiments, the second polypeptide is a recombinant polypeptideproduced by a host cell or a therapeutic polypeptide or serum albumin.In some embodiments, the second polypeptide is expressed from a vectortransfected into a host cell, such as a mammalian host cell, such as aChinese Hamster Ovary (CHO) cell.

In some embodiments, the sample is a cell culture sample or a blood or aserum sample or a pharmaceutical product or an intermediate thereof.

In some embodiments, the peptide products of the plurality ofpolypeptides in the sample are obtained via sample digestion prior toanalyzing the peptide products using the LC/MS technique. In someembodiments, the peptide products are tryptic peptide products of theplurality of polypeptides.

In some embodiments, the plurality of sample loading quantitiescomprises sample loading quantities in the range of about 0.1-25 μgtotal protein. In some embodiments, the first sample loading quantity isabout 10 μg total protein. In some embodiments, the second sampleloading quantity is about 0.5 μg to 10 μg, or 3 μg to 6 μg, or 1 μg, 2μg, 3 μg, 4 μg, 5 μg, or 6 μg total protein.

In some embodiments, the methods further comprise selecting the secondsample loading quantity based on MS signals of the second set of peptideproducts.

In some embodiments, the methods further comprise selecting the firstsample loading quantity based on MS signals of the first set of peptideproducts.

In some embodiments, each of the plurality of sample loading quantitieshas the same total volume.

In some embodiments, n is 1 or greater or n is 3.

In some embodiments, m is 1 or greater or m is 3.

In some embodiments, the middle set of qualified ions of peptideproducts of the second polypeptide are selected based on the sequencesof each of the peptide products.

In another aspect, the present invention provides methods for detectinga contaminate polypeptide in the production of a therapeuticpolypeptide, the method comprising: (a) obtaining a sample comprisingthe therapeutic polypeptide; (b) determining if the contaminatepolypeptide is present in the sample; wherein the presence of thecontaminate polypeptide is based on the absolute quantification of thecontaminant polypeptide in the sample using the quantification methodsprovided herein.

In another aspect, the present invention provides systems for absolutequantification of a first polypeptide in a sample comprising the firstpolypeptide and a second polypeptide, the system comprising: (a) a massspectrometer; (b) a computer comprising; (c) a non-transitory computerreadable medium including instructions stored thereon which, whenexecuted, perform processing including: calculating the average or sumof an MS signal for: (i) a top set of n number of qualified ions ofpeptide products of the first polypeptide with the highest MS signals atthe first sample loading quantity (A); (ii) a top set of n number ofqualified ions of peptide products of the second polypeptide with thehighest MS signals at the second sample loading quantity (B); (iii) amiddle set of m number of qualified ions of peptide products of thesecond polypeptide at the first sample loading quantity (C); and (iv)the middle set of qualified ions of peptide products of the secondpolypeptide at the second sample loading quantity (D), wherein A, B, C,and D are all calculated using the average or all calculated using thesum of the MS signal; and determining an absolute quantity of the firstpolypeptide in the first sample loading quantity based on the followingformula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof. In some embodiments, the systemsfurther comprise a liquid chromatograph.

In another aspect, the present invention provides non-transitorycomputer readable mediums including instructions stored thereon which,when executed, perform processing for absolute quantification of a firstpolypeptide in a sample comprising the first polypeptide and a secondpolypeptide, the processing including: calculating the average or sum ofan MS signal for: (i) a top set of n number of qualified ions of peptideproducts of the first polypeptide with the highest MS signals at thefirst sample loading quantity (A); (ii) a top set of n number ofqualified ions of peptide products of the second polypeptide with thehighest MS signals at the second sample loading quantity (B); (iii) amiddle set of m number of qualified ions of peptide products of thesecond polypeptide at the first sample loading quantity (C); and (iv)the middle set of qualified ions of peptide products of the secondpolypeptide at the second sample loading quantity (D), wherein A, B, C,and D are all calculated using the average or all calculated using thesum of the MS signal; and determining an absolute quantity of the firstpolypeptide in the first sample loading quantity based on the followingformula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a histogram of MS peak areas for the forty most abundantpeptide product ions observed from a LC/MS analysis of a samplecomprising sphingomyelin phosphodiesterase (ASM) at different sampleloading quantities (the sample loading quantity per LC/MS analysis isordered as 1 μg, 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg, 18 μg, and20 μg, from left to right for each peptide product bar set). The averagepercent error for each peptide product ion across the sample loadingquantities is shown above the peptide product bar set.

FIG. 2 shows the summed peak area of a middle set of three peptideproducts (Middle-3; squares) and a top set of three peptide products(Top-3; diamonds) over six concentration points. The R² for a linearregression of the Middle-3 peptide products is 0.98 and for the Top-3peptide products is 0.87.

FIGS. 3A-3B show a comparison of the Hi-3 (FIG. 3A) and the Mid-3 (FIG.3B) quantification methods for four samples (Lot 1, Lot 2, Lot 3, Lot 4,from left to right for each bar set) at four different assay occasions(Occasion A, Occasion B, Occasion C, Occasion D). There was an 82%relative standard deviation for the Hi-3 method (FIG. 3A) and a 16%relative standard deviation for the Mid-3 method (FIG. 3B).

FIG. 4 shows the relative abundance of the top 8 identified host cellproteins across various therapeutic protein production lots.

FIG. 5 shows the relative abundance of identified host cell proteinsacross stages of a purification process for a therapeutic protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for absolute quantification of afirst polypeptide in a sample comprising a plurality of polypeptidescomprising the first polypeptide and a second polypeptide, the methodscomprising determining an absolute quantity of the first polypeptide inthe sample based on the average or sum of MS signals for: a top set of nnumber of qualified ions of peptide products of the first polypeptidewith the highest MS signals at the first sample loading quantity (A); atop set of n number of qualified ions of peptide products of the secondpolypeptide with the highest MS signals at the second sample loadingquantity (B); a middle set of m number of qualified ions of peptideproducts of the second polypeptide at the first sample loading quantity(C); and the middle set of qualified ions of peptide products of thesecond polypeptide at the second sample loading quantity (D), wherein A,B, C, and D are all calculated using the average or all calculated usingthe sum of MS signals. In some embodiments, the first polypeptide isless abundant than a second polypeptide in the sample. In someembodiments, the absolute quantity of a first polypeptide is determinedby the following formula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)].

The methods of the present invention are also referred to herein asMid-3. As used herein, “qualified,” as used in reference to ions, refersto peptide product ions that are suitable for the quantification methodsdescribed herein. In some embodiments, the qualified peptide productions exclude non-tryptic peptide product ions or peptide product ionswith post-translational modifications.

The methods of the present invention described herein provide, forexample, improved accuracy and reproducibility of polypeptidequantification over a larger dynamic range of polypeptide concentrationsin a sample, as compared to label-free absolute quantification methodsknown in the art (e.g., Hi-3; J. C. Silva et al., supra). Withoutwishing to be bound by theory, label-free absolute quantificationmethods are based on the finding that, assuming an equimolar amount ofeach of a plurality of polypeptides in a sample, the average MS detectorresponse from the top n most abundant ions of a peptide product of apolypeptide is similar across each of the plurality of polypeptides(i.e., peptide product concentration correlates with detector response).Therefore, the quantity of a polypeptide of unknown concentration may bedetermined by comparison to a standard polypeptide of knownconcentration. However, using the most abundant ions of peptide productsof a polypeptide standard for absolute quantification may lead to pooraccuracy and reproducibility. For example, quantification inaccuracy mayarise due to the observation of nonlinear behavior for top ionizingpeptide products due to MS detector saturation. Furthermore, suchmethods may rely on spiking in a known quantity of a standardpolypeptide or analyzing a known quantity of a standard protein samplein a LC/MS analysis separate from the sample containing the polypeptideof unknown concentration. Both approaches may lead to a reduction inquantification accuracy and an increase in variability. For example,reproducibly aliquoting a known quantity of a standard polypeptide toany number of samples may be challenging and calibrating aquantification calculation based on a standard polypeptide that isdigested in a separate enzymatic reaction from the sample containing theunknown polypeptide may create additional variation in based on thedegree of digestion completion.

The present invention provides quantification methods that integrateelements to achieve improved accuracy and reproducibility of polypeptidequantification. First, the absolute quantification methods of thepresent invention take advantage of polypeptide sample systemscomprising a polypeptide with a known concentration and that is in highabundance relative to other polypeptides in the sample (e.g., atherapeutic protein in a manufacturing sample or albumin in a serumsample) and do not require comparison to a spiked-in or separatelyanalyzed standard polypeptide. Second, the absolute quantificationmethods of the present invention use MS measurements at twoconcentrations to avoid MS detector saturation of the top n peptideproduct ions of the high abundant polypeptide. Third, the absolutequantification methods of the present invention further utilize a middleset of peptide product ions with reduced quantification error incomparison to top peptides to reduce overall quantification error. Theadvantages of the methods of the present invention were demonstrated ina direct comparison to the label-free absolute quantification methodknown in the art (i.e., Hi-3). For example, as shown in Example 2,across a series of assays and samples the methods disclosed in thepresent invention achieved a 16% relative standard deviation, whereasthe Hi-3 method had an 82% relative standard deviation.

As discussed below in more detail, the present invention providesmethods useful for MS-based label-free absolute quantification oflow-abundance polypeptides (e.g., a first polypeptide) using informationfrom another polypeptide (e.g., the second polypeptide) that has a knownconcentration and is higher in relative abundance than the low-abundancepolypeptides, the methods including any one or more of the following:(a) performing a loading study to determine two sample loadingconcentrations at which data is obtained and/or analyzed forquantification of low-abundance polypeptides (e.g., the first sampleloading quantity and the second sample loading quantity); (b) selectingqualified peptide product ions for polypeptide quantification (e.g., atop set of n number of qualified ions of peptide products of the firstpolypeptide, a top set of n number of qualified ions of peptide productsof the second polypeptide, and a middle set of m number of qualifiedions of peptide products of the second polypeptide); and (c) determiningabsolute polypeptide quantity of a low-abundance polypeptide using MSsignals (e.g., MS signals from peptide products of the first polypeptideand the second polypeptide).

Performing a Loading Study to Determine a First Sample Loading Quantityand a Second Sample Loading Quantity

The present invention provides methods for performing a loading study ofa sample over the desired dynamic range of the assay. The MS signalinformation obtained from the loading study allows for, for example,selection of a first sample loading quantity wherein the peptideproducts of the first polypeptide are detectable (the first polypeptideis in low abundance relative to the second polypeptide), selection of asecond sample loading quantity wherein the top n number of qualifiedhighest abundant peptide product ions of the second polypeptide do notsaturate the MS detector, and selection of a first sample loadingquantity and a second sample loading quantity wherein a middle set of mnumber of qualified ions of peptide products of the second polypeptidedemonstrate reduced quantification error (i.e., increased linearbehavior relative to the most abundant peptide product ions).

In some embodiments, the methods comprise analyzing peptide products ofa plurality of polypeptides at a plurality of sample loading quantitiesusing a liquid chromatography/mass spectrometry (LC/MS) technique toobtain MS signals of ions of the peptide products of the plurality ofpolypeptides at each of the plurality of sample loading quantities,wherein the plurality of sample loading quantities comprises a firstsample loading quantity and a second sample loading quantity, andwherein the first sample loading quantity is greater than the secondsample loading quantity. In some embodiments, the MS signal isionization intensity. In some embodiments, the MS signal is peak height.In some embodiments, the MS signal is peak area. In some embodiments,the MS signal is peak volume.

In some embodiments, information obtained from a loading study may beapplied to subsequent sample analyses for the quantification of apolypeptide (e.g., selecting 2 sample loading quantities for additionalsample analyses via an LC/MS technique).

In some embodiments, the plurality of sample loading quantitiescomprises at least 2 sample loading quantities. In some embodiments, theplurality of sample loading quantities comprises 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 sample loading quantities.

Sample loading quantities may vary depending on the LC/MSinstrumentation used (e.g., based on the loading capacity of achromatography column). In some embodiments, the plurality of sampleloading quantities comprises sample loading quantities in the range ofabout 0.1 μg to about 100 μg, about 0.1 μg to about 75 μg, about 0.1 μgto about 50 μg, about 0.1 μg to about 40 μg, about 0.1 μg to about 30μg, about 0.1 μg to about 20 μg, about 0.1 μg to about 15 μg, about 0.1μg to about 10 μg, about 1 μg to about 30 μg, about 1 μg to about 20 μg,about 1 μg to about 15 μg, or about 1 μg to about 10 μg.

In some embodiments, the sample loading quantity is about 0.5 μg, about1 μg, about 1.5 μg, about 2 μg, about 2.5 μg, about 3 μg, about 3.5 μg,about 4 μg, about 4.5 μg, about 5 μg, about 5.5 μg, about 6 μg, about6.5 μg, about 7 μg, about 7.5 μg, about 8 μg, about 8.5 μg, about 9 μg,about 9.5 μg, about 10 μg, about 10.5 μg, about 11 μg, about 11.5 μg,about 12 μg, about 12.5 μg, about 13 μg, about 13.5 μg, about 14 μg,about 14.5 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg,about about 26 μg, about 27 μg, about 28 μg, about 29 μg, or about 30μg.

In some embodiments, the plurality of sample loading quantitiescomprises a first sample loading quantity and a second sample loadingquantity, wherein the first sample loading quantity is about 10 andwherein the second sample loading quantity is about 0.5 μg to 10 μg, or3 μg to 6 μg, or 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, or 6 μg.

In some embodiments, the first sample loading quantity is selected basedon MS signals of peptide products of the first polypeptide. In someembodiments, the first sample loading quantity is selected based on MSsignals of peptide products of the second polypeptide. In someembodiments, the first sample loading quantity is selected based on MSsignals of peptide products of the first polypeptide and MS signals ofpeptide products of the second polypeptide.

In some embodiments, the second sample loading quantity is selectedbased on MS signals of peptide products of the second polypeptide.

In some embodiments, subsequent sample loading quantities analyzed in aloading study are based on data from a previously analyzed sampleloading quantity.

The volume of each sample loading quantity may vary depending on theLC/MS instrumentation used (e.g., based on the size of the sample loop).In some embodiments, each of the plurality of sample loading quantitieshas the same total volume. In some embodiments, the volume of each of aplurality of sample loading quantities is about 1 μL to about 60 μL,about 10 μL to about 60 μL, about 20 μL to about 50 μL, or about 30 μLto about 50 μL. In some embodiments, the volume of each of a pluralityof sample loading quantities is the same and is about 1 μL to about 60μL, about 10 μL to about 60 μL, about 20 μL to about 50 μL, or about 30μL to about 50 μL. In some embodiments, the volume of each of aplurality of sample loading quantities is about 5 μL, 10 μL, 15 μL, 20μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, or 60 μL.

Selecting Qualified Peptide Product Ions of the First Polypeptide andthe Second Polypeptide

The present invention provides methods for selecting sets of qualifiedions of peptide products for absolute quantification of a firstpolypeptide in a sample comprising a plurality of polypeptidescomprising the first polypeptide and a second polypeptide, wherein thefirst polypeptide is in lower abundance relative to the secondpolypeptide. For example, the invention provides methods for selectionof any one of: a top set of n number of qualified ions of peptideproducts of the first polypeptide, a top set of n number of qualifiedions of peptide products of the second polypeptide, and a middle set ofm number of qualified ions of peptide products of the secondpolypeptide.

In some embodiments, the methods of the present invention compriseselecting a top set of n number of qualified ions of peptide products ofthe first polypeptide with the highest MS signal at the first sampleloading quantity.

In some embodiments, the methods of the present invention compriseselecting a top set of n number of qualified ions of peptide products ofthe second polypeptide with the highest MS signals at the second sampleloading quantity.

The number of MS identifiable peptide products of a polypeptide may varydepending on concentration and characteristics of the polypeptide. Insome embodiments, the concentration and characteristics of the firstpolypeptide may result in identification of a limited number of peptideproducts at the first sample loading quantity. In some embodiments, n is1 or greater, wherein n is an integer. In some embodiments, n is 3. Insome embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the qualified ions of peptide products with thehighest MS signals may exclude peptide product ions with certaincharacteristics, including, for example, missed enzymatic cleavages,post-translational modifications, and overlapping LC elution profilesand isotope distributions. For example, if a sample is digested withtrypsin and the peptide product with the highest MS signal is anon-tryptic peptide product, this peptide product may be excluded fromthe selection of a top set of n number of qualified ions of peptideproducts of the first polypeptide or a top set of n number of qualifiedions of peptide products of the second polypeptide. In some embodiments,the qualified ions of a peptide product with the highest MS signals mayexclude peptide product ions originating from portions of a polypeptidethat are not reproducibly present as part of the originating polypeptide(e.g., the peptide product originating from a cleavage product of thepolypeptide and thus may not be present in the sample at the sameconcentration as the originating polypeptide).

In some embodiments, the methods of the present invention compriseselecting a middle set of m number of qualified ions of peptide productsof the second polypeptide, wherein the middle set of qualified ions ofpeptide products of the second polypeptide is selected based onquantification error of the qualified ions of peptide products of thesecond polypeptide from the plurality of sample loading quantities, orthe first sample loading quantity and/or the second sample loadingquantity. Generally, each peptide product ion of the middle set of mnumber of qualified ions of peptide products of the second polypeptideis selected based on having the lowest quantification error relative tothe total set of ions of peptide products of the second polypeptide. Insome embodiments, each peptide product ion of the middle set of m numberof qualified ions of peptide products of the second polypeptide has alower quantification error than the most abundant peptide product ion ofthe second polypeptide. In some embodiments, peptide product ions of themiddle set of m number of qualified ions of peptide products of a secondpolypeptide have lower quantification error than a top set of n numberof qualified ions of peptide products of the second polypeptide with thehighest MS signals.

In some embodiments, the qualified ions of peptide products with thelowest quantification error may exclude peptide product ions withcertain characteristics, including, for example, missed enzymaticcleavages, post-translational modifications, and overlapping LC elutionprofiles and isotope distributions. For example, in some embodiments, ifa sample is digested with trypsin and the peptide product with thelowest quantification error is a non-tryptic peptide product, thispeptide product may be excluded from the selection of a middle set of mnumber of qualified ions of peptide products of the second polypeptide.In some embodiments, the qualified ions of a peptide product with thehighest MS signals may exclude peptide product ions originating fromportions of a polypeptide that are not reproducibly present as part ofthe originating polypeptide (e.g., the peptide product originating froma cleavage product of the polypeptide and thus may not be present in thesample at the same concentration as the originating polypeptide).

In some embodiments, selection of a middle set of m number of qualifiedions of peptide products of the second polypeptide is based onquantification error obtained from a loading study. In some embodiments,selection of a middle set of m number of qualified ions of peptideproducts of the second polypeptide is based on quantification errorobtained from a plurality of sample loading quantities. In someembodiments, quantification error is an average percent error of aplurality of sample loading quantities. In some embodiments, selectionof a middle set of m number of qualified ions of peptide products of thesecond polypeptide is based on quantification error obtained from afirst sample loading quantity. In some embodiments, selection of amiddle set of m number of qualified ions of peptide products of thesecond polypeptide is based on quantification error obtained from asecond sample loading quantity. In some embodiments, selection of amiddle set of m number of qualified ions of peptide products of thesecond polypeptide is based on quantification error obtained from afirst sample loading quantity and a second sample loading quantity.

In some embodiments, m is 1 or greater, wherein m is an integer. In someembodiments, m is 3. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8,9, or 10.

In some embodiments, n is equal to m. In some embodiments, n is notequal to m. In some embodiments, n and m are 2 or greater, wherein n andm are an integer. In some embodiments, n and m are 3.

In some embodiments, each of the top set of qualified ions of peptideproducts of a second polypeptide is different than each of a middle setof qualified ions of peptide products of the second polypeptide. In someembodiments, a qualified peptide product ion is a member of a top set ofqualified ions of peptide products of a second polypeptide and a middleset of qualified ions of peptide products of the second polypeptide.

In some embodiment, the qualified peptide product ion comprises acarbamidomethylated cysteine. In some embodiment, the qualified peptideproduct ion comprises a carboxymethylated cysteine.

Determining Absolute Polypeptide Quantity of Low-Abundance Polypeptides

The present invention provides methods for calculating the absolutepolypeptide quantity of a first polypeptide in a sample comprising aplurality of polypeptides comprising the first polypeptide and a secondpolypeptide, the methods comprising determining an absolute quantity ofthe first polypeptide in the sample based on the average or sum of MSsignals for: a top set of n number of qualified ions of peptide productsof the first polypeptide with the highest MS signals at the first sampleloading quantity (A); a top set of n number of qualified ions of peptideproducts of the second polypeptide with the highest MS signals at thesecond sample loading quantity (B); a middle set of m number ofqualified ions of peptide products of the second polypeptide at thefirst sample loading quantity (C); and the middle set of qualified ionsof peptide products of the second polypeptide at the second sampleloading quantity (D), wherein A, B, C, and D are all calculated usingthe average or all calculated using the sum of MS signals.

In some embodiments, the absolute quantity of a first polypeptide isdetermined based on the following formula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof.

In some embodiments, A, B, C, and D are all calculated using the averageof MS signals. In some embodiments, A, B, C, and D are all calculatedusing the sum of MS signals.

In some embodiments, the MS signal is ionization intensity. In someembodiments, the MS signal is peak height. In some embodiments, the MSsignal is peak area. In some embodiments, the MS signal is peak volume.

In some embodiments, the top set of n number of qualified ions ofpeptide products of a first polypeptide with the highest MS signals ispredetermined. In some embodiments, the top set of n number of qualifiedions of peptide products of a second polypeptide with the highest MSsignals is predetermined. In some embodiments, the middle set of mnumber of qualified ions of peptide products of a second polypeptide ispredetermined. In some embodiments, the ratio of a top set of n numberof qualified ions of peptide products of a second polypeptide with thehighest MS signals at a second sample loading quantity (B) and a middleset of qualified ions of peptide products of the second polypeptide atthe second sample loading quantity (D) is predetermined.

In some embodiments, the average or sum of MS signals for: a top set ofn number of qualified ions of peptide products of the first polypeptidewith the highest MS signals at the first sample loading quantity (A); atop set of n number of qualified ions of peptide products of the secondpolypeptide with the highest MS signals at the second sample loadingquantity (B); a middle set of m number of qualified ions of peptideproducts of the second polypeptide at the first sample loading quantity(C); and the middle set of qualified ions of peptide products of thesecond polypeptide at the second sample loading quantity (D), aredetermined from 2 LC/MS analyses of the sample. In some embodiments,additional replicates analyses of the 2 LC/MS analyses are performed.

In some embodiments, the average or sum of MS signals for: a top set ofn number of qualified ions of peptide products of the first polypeptidewith the highest MS signals at the first sample loading quantity (A); atop set of n number of qualified ions of peptide products of the secondpolypeptide with the highest MS signals at the second sample loadingquantity (B); a middle set of m number of qualified ions of peptideproducts of the second polypeptide at the first sample loading quantity(C); and the middle set of qualified ions of peptide products of thesecond polypeptide at the second sample loading quantity (D), aredetermined from 2 LC/MS analyses of the sample, wherein the LC/MSanalyses are not part of the loading study. In some embodiments,additional replicates analyses of the 2 LC/MS analyses are performed.

In some embodiments, the quantification method comprises MS signals fromtwo or more polypeptides of known quantity.

In some embodiments, the methods further comprise determining theabsolute quantity of a second polypeptide. Methods for determiningprotein quantity of a second polypeptide include, for example, ELISA andWestern blot.

It is contemplated that more than one polypeptide of unknownconcentration per assay can be identified and quantified using themethods of the present invention. For example, from the equationdisclosed above, the top set of n number of qualified ions of peptideproducts of the first polypeptide with the highest MS signals at thefirst sample loading quantity (A) can be substituted with a top set of nnumber of qualified ions of peptide products of another polypeptide withthe highest MS signals at the first sample loading quantity to calculatethe quantity of the other polypeptide.

Samples and Sample Preparation

The methods of the present invention are useful for absolutequantification of a first polypeptide in a sample comprising a pluralityof polypeptides comprising the first polypeptide and a secondpolypeptide, wherein the first polypeptide is in lower abundancerelative to the second polypeptide.

In some embodiments, the first polypeptide is at least about 10-foldlower in abundance than a second polypeptide. In some embodiments, thefirst polypeptide is at least about 100-fold lower in abundance than asecond polypeptide. In some embodiments, the first polypeptide is atleast about 1000-fold lower in abundance than a second polypeptide. Insome embodiments, the first polypeptide is at least about 2-fold toabout 1×10⁹-fold lower in abundance than a second polypeptide. Forexample, the first polypeptide is measured at a quantity of one part perbillion.

In some embodiments, the second polypeptide is at least about 10-foldgreater in abundance than a first polypeptide. In some embodiments, thesecond polypeptide is at least about 100-fold greater in abundance thana first polypeptide. In some embodiments, the second polypeptide is atleast about 1000-fold greater or 1×10⁹-fold greater in abundance than afirst polypeptide. In some embodiments, the second polypeptide is atleast about 2-fold to about 1×10⁹-fold higher in abundance than a firstpolypeptide.

Sample preparation techniques necessary to produce peptide products of aplurality of polypeptides in a sample for analysis via LC/MS techniquesare known in the art.

In some embodiments, the peptide products of a plurality of polypeptidesin a sample are obtained via sample digestion prior to analyzing thepeptide products using an LC/MS technique. In some embodiments, sampledigestion comprises enzymatic digestion using a protease. In someembodiments, the enzymatic digestion is performed using one or more oftrypsin, Lys-C, IdeS, IdeZ, PNGase F, thermolysin, pepsin, elastase,Arg-C, TEV, Glu-C, Asp-N, and Factor Xa. In some embodiments, thepeptide products of a plurality of polypeptides in a sample are trypticpeptide products.

In some embodiments, sample digestion comprises chemical digestion, suchas acid hydrolysis.

In some embodiments, the methods comprise obtaining a sample. Techniquesfor obtaining samples for LC/MS analysis are known in the art andinclude, for example, tissue (e.g., blood, plasma) collection and cellculture.

In some embodiments, the sample has been purified or enriched. In someembodiments, the methods comprise processing the plurality ofpolypeptides in a sample to produce peptide products. In someembodiments, processing the plurality of polypeptides in a samplecomprises one or more of: (a) centrifuging the sample to isolate theplurality of polypeptides; (b) purifying the plurality of polypeptidesin the sample; (c) removing from the sample components incompatible withsubsequent processing and LC/MS analysis; (d) digesting the plurality ofpolypeptides to produce the peptide products; and (e) purifying thepeptide products prior to LC/MS analysis.

In some embodiments, the first polypeptide is a host cell protein. Insome embodiments, the second polypeptide is a recombinant polypeptideproduced by a host cell. In some embodiments, the second polypeptide isa therapeutic polypeptide, such as an antibody (e.g., a recombinantantibody), or an enzyme (e.g., a recombinant enzyme), or a peptide(e.g., an insulin). In some embodiments, the second polypeptide is viralprotein, such as a capsid of a viral particle (e.g., such as for a genetherapy). In some embodiments, the sample is a cell culture sample. Insome embodiments, the sample is a pharmaceutical product or anintermediate thereof.

In some embodiments, the first polypeptide is a biomarker, such acirculating biomarker. In some embodiments, the second polypeptide isserum albumin. In some embodiments, the sample is a blood or serumsample.

Liquid Chromatography/Mass Spectrometry (LC/MS) Techniques

The present invention contemplates a diverse array of LC/MS techniquesfor generating tandem mass spectra of a sample comprising a plurality ofpolypeptides comprising the first polypeptide and a second polypeptide.

In some embodiments, the LC/MS technique comprises separating thepeptide products via a liquid chromatography technique. Liquidchromatography techniques contemplated by the present applicationinclude methods for separating polypeptides and liquid chromatographytechniques compatible with mass spectrometry techniques. In someembodiments, the liquid chromatography technique comprises a highperformance liquid chromatography technique. Thus, in some embodiments,the liquid chromatography technique comprises an ultra-high performanceliquid chromatography technique. In some embodiments, the liquidchromatography technique comprises a high-flow liquid chromatographytechnique. In some embodiments, the liquid chromatography techniquecomprises a low-flow liquid chromatography technique, such as amicro-flow liquid chromatography technique or a nano-flow liquidchromatography technique. In some embodiments, the liquid chromatographytechnique comprises an online liquid chromatography technique coupled toa mass spectrometer. In some embodiments, the online liquidchromatography technique is a high performance liquid chromatographytechnique. In some embodiments, the online liquid chromatographytechnique is an ultra-high performance liquid chromatography technique.

In some embodiments, capillary electrophoresis (CE) techniques, orelectrospray or MALDI techniques may be used to introduce the sample tothe mass spectrometer.

In some embodiment, the mass spectrometry technique comprises anionization technique. Ionization techniques contemplated by the presentapplication include techniques capable of charging polypeptides andpeptide products. Thus, in some embodiments, the ionization technique iselectrospray ionization. In some embodiments, the ionization techniqueis nano-electrospray ionization. In some embodiments, the ionizationtechnique is atmospheric pressure chemical ionization. In someembodiments, the ionization technique is atmospheric pressurephotoionizationionization. In some embodiments, the ionization techniqueis matrix-assisted laser desorption ionization (MALDI). In someembodiment, the mass spectrometry technique comprises electrosprayionization, nanoelectrospray ionization, or a matrix-assisted laserdesorption ionization (MALDI) technique.

In some embodiments, the LC/MS technique comprises analyzing the peptideproducts via a mass spectrometry technique. Mass spectrometerscontemplated by the present invention, to which an online liquidchromatography technique is coupled, include high-resolution massspectrometers and low-resolution mass spectrometers. Thus, in someembodiments, the mass spectrometer is a time-of-flight (TOF) massspectrometer. In some embodiments, the mass spectrometer is a quadrupoletime-of-flight (Q-TOF) mass spectrometer. In some embodiments, the massspectrometer is a quadrupole ion trap time-of-flight (QIT-TOF) massspectrometer. In some embodiments, the mass spectrometer is an ion trap.In some embodiments, the mass spectrometer is a single quadrupole. Insome embodiments, the mass spectrometer is a triple quadrupole (QQQ). Insome embodiments, the mass spectrometer is an orbitrap. In someembodiments, the mass spectrometer is a quadrupole orbitrap. In someembodiments, the mass spectrometer is a fourier transform ion cyclotronresonance (FT) mass spectrometer. In some embodiments, the massspectrometer is a quadrupole fourier transform ion cyclotron resonance(Q-FT) mass spectrometer. In some embodiments, the mass spectrometrytechnique comprises positive ion mode. In some embodiments, the massspectrometry technique comprises negative ion mode. In some embodiments,the mass spectrometry technique comprises a time-of-flight (TOF) massspectrometry technique. In some embodiments, the mass spectrometrytechnique comprises a quadrupole time-of-flight (Q-TOF) massspectrometry technique. In some embodiments, the mass spectrometrytechnique comprises an ion mobility mass spectrometry technique. In someembodiments a low-resolution mass spectrometry technique, such as an iontrap, or single or triple-quadrupole approach is appropriate.

In some embodiments, the LC/MS technique comprises processing theobtained MS signals of the peptide products. In some embodiments, theLC/MS technique comprises peak detection. In some embodiments, the LC/MStechnique comprises determining ionization intensity of a peptideproduct. In some embodiments, the LC/MS technique comprises determiningpeak height of a peptide product. In some embodiments, the LC/MStechnique comprises determining peak area of a peptide product. In someembodiments, the LC/MS technique comprises determining peak volume of apeptide product. In some embodiments, the LC/MS technique comprisesidentifying the peptide products by amino acid sequence. In someembodiments, the LC/MS technique comprises manually validating thepeptide product amino acid sequence assignments. In some embodiments,the LC/MS technique comprises identifying the first polypeptide by aprotein identifier. In some embodiments, the LC/MS technique comprisesidentifying one or more of the plurality of polypeptides by a proteinidentifier, which may be identified in a database search or a librarysearch.

In some embodiments, identification of peptide products of a polypeptidecan be achieved using spectral libraries. Generally, use of spectrallibraries allows for the imputation of knowledge gained regarding apolypeptide system and results in increased speed of data analysis anddecreased error.

Use of Absolute Quantification Methods

The MS-based label-free absolute quantification methods disclosed hereinare especially suited for uses comprising quantification of alow-abundance polypeptide in a sample comprising the low-abundancepolypeptide and another polypeptide of relative high-abundance. TheMS-based label-free absolute quantification methods disclosed hereinmay, e.g., constitute a single step in a multi-step process, such asquantification of a low-abundance protein in the purification of atherapeutic protein.

In some embodiments, the present invention provides methods of detectinga contaminate polypeptide in the production of a therapeuticpolypeptide, the methods comprising: (a) obtaining a sample comprisingthe therapeutic polypeptide; (b) determining if the contaminatepolypeptide is present in the sample, wherein the presence of thecontaminate polypeptide is based on the absolute quantification of thecontaminate polypeptide in the sample using the methods disclosedherein. In some embodiments, the contaminate polypeptide is a host cellprotein. In some embodiments, the contaminate polypeptide is a viralprotein, such as a capsid protein. In some embodiments, the secondpolypeptide is a therapeutic polypeptide. In some embodiments, more thanone contaminate polypeptide is detected in a sample (e.g., and the totalamount of contaminate polypeptides in the sample is quantified). In oneembodiment, the sample is taken at various steps during the productionprocess of a recombinant polypeptide (e.g., a therapeutic polypeptide),to assay for purity of the recombinant polypeptide at the various steps.The lower the amount of contaminate polypeptides (e.g., host cellpolypeptides) identified, will indicate the higher the purity of therecombinant polypeptide.

In some embodiments, the present invention provides methods of producinga therapeutic polypeptide, the methods comprising: (a) obtaining asample comprising the therapeutic polypeptide from a stage of theproduction process, e.g., cell culture harvest or a purification step;(b) identifying a contaminate polypeptide in the sample; (c) determiningthe level of the contaminate polypeptide in the sample, wherein thelevel of the contaminate polypeptide is based on the absolutequantification of the contaminate polypeptide in the sample using themethods disclosed herein. In some embodiments, the contaminatepolypeptide is a host cell protein. In some embodiments, the contaminatepolypeptide is a viral protein, such as a capsid protein. In someembodiments, the second polypeptide is a therapeutic polypeptide. Insome embodiments, more than one contaminate polypeptide is detected in asample (e.g., and the total amount of contaminate polypeptides in thesample is quantified). In one embodiment, the sample is taken at varioussteps during the production process of a recombinant polypeptide (e.g.,a therapeutic polypeptide), to assay for purity of the recombinantpolypeptide at the various steps. The lower the amount of contaminatepolypeptides (e.g., host cell polypeptides) identified, will indicatethe higher the purity of the recombinant polypeptide.

In some embodiments, the present invention provides methods of purifyinga therapeutic polypeptide, the methods comprising: (a) obtaining asample comprising the therapeutic polypeptide from one or more stages ofa purification process, e.g., cell culture harvest or a purificationstep; (b) identifying a contaminate polypeptide in the sample; (c)determining the level of the contaminate polypeptide in the sample atthe one or more stages of a purification process, wherein the level ofthe contaminate polypeptide is based on the absolute quantification ofthe contaminate polypeptide in the sample using the methods disclosedherein. In some embodiments, the contaminate polypeptide is a host cellprotein. In some embodiments, the contaminate polypeptide is a viralprotein, such as a capsid protein. In some embodiments, the secondpolypeptide is a therapeutic polypeptide. In some embodiments, more thanone contaminate polypeptide is detected in a sample (e.g., and the totalamount of contaminate polypeptides in the sample is quantified). In oneembodiment, the sample is taken at various steps during the productionprocess of a recombinant polypeptide (e.g., a therapeutic polypeptide),to assay for purity of the recombinant polypeptide at the various steps.The lower the amount of contaminate polypeptides (e.g., host cellpolypeptides) identified, will indicate the higher the purity of therecombinant polypeptide.

In some embodiments, the present invention provides methods of detectinga contaminate polypeptide in a gene therapy product. The methods includefor example, (a) obtaining a sample comprising a gene therapy vector,from one or more stages of a purification process, e.g., cell cultureharvest or a purification step; (b) identifying a contaminatepolypeptide in the sample; (c) determining the level of the contaminatepolypeptide in the sample at the one or more stages of a purificationprocess, wherein the level of the contaminate polypeptide is based onthe absolute quantification of the contaminate polypeptide in the sampleusing the methods disclosed herein. In some embodiments, the genetherapy product is associated with a viral protein, such as a capsid. Insome embodiments, the gene therapy product comprises a viral protein,such as a capsid. In some embodiments, the gene therapy vector isassociated with a viral protein, such as a capsid. In some embodiments,the gene therapy vector is part of a viral particle comprising a viralprotein, such as a capsid. In some embodiments, the contaminatepolypeptide is a host cell protein. In some embodiments, the contaminatepolypeptide is a viral protein, such as a capsid protein. In someembodiments, the contaminate polypeptide is a viral protein that is notassociated with the gene therapy product. In some embodiments, thecontaminate polypeptide is a viral protein that is not a part of thegene therapy product. In some embodiments, the contaminate polypeptideis a helper virus protein. In some embodiments, the second polypeptideis a viral protein, such as a capsid protein. In some embodiments, morethan one contaminate polypeptide is detected in a sample (e.g., and thetotal amount of contaminate polypeptides in the sample is quantified).In one embodiment, the sample is taken at various steps during theproduction process of the gene therapy product, to assay for purity ofthe gene therapy product at the various steps. The lower the amount ofcontaminate polypeptides (e.g., host cell polypeptides) identified, willindicate the higher the purity of the gene therapy product.

In some embodiments, the present invention provides methods of producinga gene therapy product. The methods include for example, (a) obtaining asample comprising a gene therapy vector, from one or more stages of apurification process, e.g., cell culture harvest or a purification step;(b) identifying a contaminate polypeptide in the sample; (c) determiningthe level of the contaminate polypeptide in the sample at the one ormore stages of a purification process, wherein the level of thecontaminate polypeptide is based on the absolute quantification of thecontaminate polypeptide in the sample using the methods disclosedherein. In some embodiments, the gene therapy product is associated witha viral protein, such as a capsid. In some embodiments, the gene therapyproduct comprises a viral protein, such as a capsid. In someembodiments, the gene therapy vector is associated with a viral protein,such as a capsid. In some embodiments, the gene therapy vector is partof a viral particle comprising a viral protein, such as a capsid. Insome embodiments, the contaminate polypeptide is a host cell protein. Insome embodiments, the contaminate polypeptide is a viral protein, suchas a capsid protein. In some embodiments, the contaminate polypeptide isa viral protein that is not associated with the gene therapy product. Insome embodiments, the contaminate polypeptide is a viral protein that isnot a part of the gene therapy product. In some embodiments, thecontaminate polypeptide is a helper virus protein. In some embodiments,the second polypeptide is a viral protein, such as a capsid protein. Insome embodiments, more than one contaminate polypeptide is detected in asample (e.g., and the total amount of contaminate polypeptides in thesample is quantified). In one embodiment, the sample is taken at varioussteps during the production process of the gene therapy product, toassay for purity of the gene therapy product at the various steps. Thelower the amount of contaminate polypeptides (e.g., host cellpolypeptides) identified, will indicate the higher the purity of thegene therapy product.

In some embodiments, the present invention provides methods of purifyinga gene therapy product. The methods include for example, (a) obtaining asample comprising a gene therapy vector, from one or more stages of apurification process, e.g., cell culture harvest or a purification step;(b) identifying a contaminate polypeptide in the sample; (c) determiningthe level of the contaminate polypeptide in the sample at the one ormore stages of a purification process, wherein the level of thecontaminate polypeptide is based on the absolute quantification of thecontaminate polypeptide in the sample using the methods disclosedherein. In some embodiments, the gene therapy product is associated witha viral protein, such as a capsid. In some embodiments, the gene therapyproduct comprises a viral protein, such as a capsid. In someembodiments, the gene therapy vector is associated with a viral protein,such as a capsid. In some embodiments, the gene therapy vector is partof a viral particle comprising a viral protein, such as a capsid. Insome embodiments, the contaminate polypeptide is a host cell protein. Insome embodiments, the contaminate polypeptide is a viral protein, suchas a capsid protein. In some embodiments, the contaminate polypeptide isa viral protein that is not associated with the gene therapy product. Insome embodiments, the contaminate polypeptide is a viral protein that isnot a part of the gene therapy product. In some embodiments, thecontaminate polypeptide is a helper virus protein. In some embodiments,the second polypeptide is a viral protein, such as a capsid protein. Insome embodiments, more than one contaminate polypeptide is detected in asample (e.g., and the total amount of contaminate polypeptides in thesample is quantified). In one embodiment, the sample is taken at varioussteps during the production process of the gene therapy product, toassay for purity of the gene therapy product at the various steps. Thelower the amount of contaminate polypeptides (e.g., host cellpolypeptides) identified, will indicate the higher the purity of thegene therapy product.

In some embodiments, the methods disclosed herein further compriseadjusting a protocol based on the presence of a contaminant polypeptide.For example, a purification process can be adjusted based on thepresence of an identified and quantified contaminant polypeptide. Suchadjustments provide methods for improving purity of a targetpolypeptide, such as a therapeutic polypeptide.

In some embodiments, the present invention provides methods of treatinga disease in an individual, wherein the individual is selected fortreatment based on an amount of a biomarker in the individual. In someembodiments, the biomarker is quantified in a serum sample. In someembodiments, the first polypeptide is a biomarker. In some embodiments,the second polypeptide is serum albumin.

In some embodiments, the present invention provides methods of assessinga disease in an individual, wherein the individual is assessed based onan amount of a biomarker in the individual. In some embodiments, theamount of the biomarker is quantified in a serum sample. In someembodiments, the first polypeptide is a biomarker. In some embodiments,the second polypeptide is serum albumin.

In some embodiments, the present invention provides methods ofdiagnosing a disease in an individual, wherein the individual isdiagnosed with the disease based on an amount of a biomarker in theindividual. In some embodiments, the amount of the biomarker isquantified in a serum sample. In some embodiments, the first polypeptideis a biomarker. In some embodiments, the second polypeptide is serumalbumin.

Systems

The present invention provides systems and non-transitory computerreadable mediums useful for determining the absolute polypeptidequantity of a first polypeptide in a sample comprising a plurality ofpolypeptides comprising the first polypeptide and a second polypeptide.

In some embodiments, the present invention provides a system forabsolute quantification of a first polypeptide in a sample comprisingthe first polypeptide and a second polypeptide, the system comprising:(a) a mass spectrometer; (b) a computer comprising; (c) a non-transitorycomputer readable medium including instructions stored thereon which,when executed, perform processing including: calculating the average orsum of an MS signal for: (i) a top set of n number of qualified ions ofpeptide products of the first polypeptide with the highest MS signals atthe first sample loading quantity (A); (ii) a top set of n number ofqualified ions of peptide products of the second polypeptide with thehighest MS signals at the second sample loading quantity (B); (iii) amiddle set of m number of qualified ions of peptide products of thesecond polypeptide at the first sample loading quantity (C); and (iv)the middle set of qualified ions of peptide products of the secondpolypeptide at the second sample loading quantity (D), wherein A, B, C,and D are all calculated using the average or all calculated using thesum of the MS signal; and determining an absolute quantity of the firstpolypeptide in the first sample loading quantity based on the followingformula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof. In some embodiments, the systemfurther comprises a liquid chromatograph.

In some embodiments, the present invention provides a non-transitorycomputer readable medium including instructions stored thereon which,when executed, perform processing for absolute quantification of a firstpolypeptide in a sample comprising the first polypeptide and a secondpolypeptide, the processing including: calculating the average or sum ofan MS signal for: (i) a top set of n number of qualified ions of peptideproducts of the first polypeptide with the highest MS signals at thefirst sample loading quantity (A); (ii) a top set of n number ofqualified ions of peptide products of the second polypeptide with thehighest MS signals at the second sample loading quantity (B); (iii) amiddle set of m number of qualified ions of peptide products of thesecond polypeptide at the first sample loading quantity (C); and (iv)the middle set of qualified ions of peptide products of the secondpolypeptide at the second sample loading quantity (D), wherein A, B, C,and D are all calculated using the average or all calculated using thesum of the MS signal; and determining an absolute quantity of the firstpolypeptide in the first sample loading quantity based on the followingformula:

[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)],

or mathematical equivalents thereof.

Those skilled in the art will recognize that several embodiments arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

As used herein, the term “polypeptide” refers to a polymer comprisingamino acids covalently joined via peptide bonds. In some embodiments,the polypeptide is a protein. In some instances, a protein comprises twoor more polypeptides (e.g., a multimeric protein, a homomeric protein, amultiprotein complex). Polypeptides may be further modified withnon-amino acid moieties. For example, a polypeptide may further compriseenzymatically-mediated modifications and/or chemical modifications(e.g., acetylation, phosphorylation, ubiquitination, formylation,glycosylation, oxidation). Such modifications may occur, for example, incell-based environments or as a result of sample processing and/oranalysis techniques.

As used herein, the term “peptide product” refers to a polymercomprising two or more amino acids covalently joined via peptide bondsobtained following decomposition processing of a polypeptide. Forexample, peptide products are obtained following decompositionprocessing of a polypeptide including chemical digestion (e.g., acidhydrolysis) or enzymatic digestion (e.g., trypsin digestion). In someembodiments, the peptide product is a tryptic peptide. In someembodiments, the peptide product is a terminal fragment of a largerpolypeptide. In some embodiments, the peptide product is an internalfragment of a polypeptide.

The term “comprises” is used herein to mean that other components,ingredients, steps, etc. are optionally present. For example, an article“comprising” components A, B, and C can consist of (i.e., contain only)components A, B, and C, or can contain not only components A, B, and Cbut also one or more other components. It is understood that “comprises”and grammatical equivalents thereof include “consisting of” or“consisting essentially of.”

Where a range of value is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictate otherwise, between the upper and lower limitof that range and any other stated or intervening value in that statedrange, is encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

EXAMPLES Example 1 Loading Study of ASM Demonstrating the ImprovedLinear MS Signal Response of a Middle Set of Peptide Product Ions of ASM

This example demonstrates a loading study using a sample comprisingsphingomyelin phosphodiesterase (ASM) for the selection of a firstsample loading quantity and a second loading quantity. Furthermore, thisexample demonstrates the improved linear MS signal response of a middleset of peptide product ions of ASM as compared to a top set of highestabundant peptide product ions of ASM.

In triplicate, an ASM sample was denatured, reduced, and alkylated priorto digestion with LysC and trypsin. LC/MS analysis was performed on thedigested samples at 1 μg, 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg,18 μg, and 20 μg. Chromatography was performed with an ACQUITY UPLC witha 2.1 by 150 mm column packed with CSH130 C18 1.7 μm material. Data wereacquired using alternating scans of low and elevated collision energy ona Xevo Q-Tof G2-XS to generate precursor and fragment ion information.MS peak areas and the average percent error for each peptide product ionover the range of sample quantities of the top 40 most abundant peptideproduct ions of ASM were calculated.

FIG. 1 shows a histogram of MS peak areas for the forty most abundantpeptide product ions observed from a LC/MS analysis of a samplecomprising sphingomyelin phosphodiesterase (ASM) at different sampleloading quantities (the sample loading quantity per LC/MS analysis isordered as 1 μg, 2.5 μg, 5 μg, 7.5 μg, 10 μg, 12.5 μg, 15 μg, 18 μg, and20 μg, from left to right for each peptide bar set). The average percenterror for each peptide product ion across the sample loading quantitiesis shown above the peptide bar set.

As shown in FIG. 1, the most intense peptide products of ASM have ahigher error relative to all peptide product ions, especially as thetotal sample quantity increases. The peptides in the middle of thepeptide product ion distribution behave more linearly and have a lowererror relative to all peptide product ions, even up to total sampleloading quantities of 20 μg.

Using data in the loading study, a top set of n number of qualified ionsof peptide products of the second polypeptide and a middle set of mnumber of qualified ions of peptide products of the second polypeptidemay be selected. For example, the top set of n number, for example, 3,of qualified ions of peptide products of ASM may include peptide productions selected from Peptide A (z=4), Peptide B (z=3), and Peptide C(z=4). The middle set of m number, for example, 3, of qualified ions ofpeptide products of ASM may include peptide product ions selected fromPeptide D (z=2), Peptide M (z=4), Peptide 0 (z=1), Peptide M (z=3),Peptide B (z=2), Peptide L (z=2), Peptide P (z=1), and Peptide Q (z=1).

Furthermore, using the data collected from the ASM loading study, thesummed peak area versus sample loaded on column (μg) was plotted foreach sample loading quantity using the top set of the 3 most abundantpeptide product ions (Top-3) and a middle set of 3 peptide product ions(Middle-3) (FIG. 2). The nonlinear behavior of the Top-3 peptide productset can be seen well below the optimal sample loading quantity (10 μg).The Middle-3 peptide product set behaved linearly over the sampleloading quantities of the loading study, thus demonstrating that theMiddle-3 peptide product ions are suitable for quantification techniquesof the present invention. The R² for a linear regression is alsoimproved using the Middle-3 peptide product set (0.98 for Middle-3peptide product set compared to 0.87 for Top-3 peptide product set).

The percent error for each peptide product set at each concentrationrelative to the 2.5 μg load is provided in Table 1. The percent errorbetween the expected ratio and observed ratio for the Middle-3 peptideproduct set was lower than the Top-3 peptide product set for all sampleloading quantities.

TABLE 1 Percent error of the Top-3 peptide product set and Middle-3peptide product set at each sample loading quantity relative to the 2.5μg load peptide product set. Load (μg) Hi-3 Error Mid-3 Error 20 76% 33%18 73% 26% 15 68% 17% 12.5 61%  5% 10 53%  5% 7.5 42% 12% 5 27% 13%

Example 2 Methods of Determining Quantity for Polypeptides

This example demonstrates the improvement of quantificationreproducibility of the Mid-3 method as compared to the Hi-3 method usingfour protein production lots of a therapeutic protein product assayed onfour different occasions.

Four protein production lots (Lot 1, Lot 2, Lot 3, Lot 4) of protein Xwere prepared and analyzed as disclosed in Example 1. For the Hi-3method, 200 fmol of a ClpB standard (E. coli Chaperone ClpB) peptideproducts was spiked in each sample as an internal standard.

As measured by the Hi-3 method, the total ppm of host cell proteinrelative to the spiked ClpB peptide products was plotted for eachprotein production lot (FIG. 3A). The quantification measurements of theHi-3 method have 82% relative standard deviation.

As measured by the Mid-3 method, the total ppm of host cell protein(HCP) was plotted for each protein production lot (FIG. 3B). Thequantification measurements of the Mid-3 method have 16% relativestandard deviation.

The equation used to calculate polypeptide quantity for the Mid-3 methodis as follows:

[(Sum of peak area of top 3 peptide product ions of HCP at the highsample quantity load)/(Sum of peak area of middle 3 peptide product ionsof the therapeutic protein product at the high sample quantityload)]*(fmol of the therapeutic protein product at high sample quantityload)*[(Sum of peak area of middle 3 peptide product ions of thetherapeutic protein product at the low sample quantity load)/(Sum ofpeak area of top 3 peptide product ions of the therapeutic proteinproduct at the low sample quantity load)].

Using the Hi-3 method with 200 fmol of ClpB peptides as the internalstandard yielded higher error, as compared to the Mid-3 method. Absolutequantitation was much more reproducible using the Mid-3 method. Thebiggest variability was due to the spiked-in internal standard, althoughdifferences in enzymatic digestion may have also occurred. At 10 μgon-column, the middle set of peptide products were more appropriate touse for quantification as they behaved linearly.

Example 3 Application of Mid-3 Quantification Method for Detection andTracking of Host Cell Proteins During Purification of a TherapeuticProtein

This example demonstrates an application of the Mid-3 quantificationmethod disclosed herein for high-throughput detection and tracking ofcontaminant host cell proteins (HCPs) at multiple stages during apurification process of a therapeutic protein, including samplescollected from a cell culture harvest to samples collected following afinal desalting protocol. This example further demonstrates use ofsoftware for tracking peptides of HCPs by retention time and m/z forlabel-free quantitation of HCPs in late-stage purification samples, anduse of spectral library-based searches to further improve throughput andoptimize absolute quantitation.

Methods:

Following cell culture-based production of a therapeutic protein(protein X), samples were collected from the cell culture harvest and at5 stages of a protein purification process, including final drugsubstance samples. Using triplicates provided statistical power ofdownstream measurements, including reproducibility and yield data. Afterthe samples were collected, harvest samples were filtered through a 3 kMWCO Amicon Ultra-15 filter (EMD Millipore) according to themanufacturer's instructions to concentrate and remove additives thatimpede mass spectrometric analysis before being denatured and digestedwith the rest of the samples. Briefly, a water rinse step of Amicondevices was completed and then 400 μg total protein or therapeuticprotein per sample was added with the 50 mM ammonium bicarbonate (Ambic)added on top to a total volume of 15 mL. Subsequently, a wash of 15 mLof 50 mM Ambic was completed, prior to a final centrifugation step.Final concentrations were measured as between 1.4 and 1.7 mg/mL totalprotein or therapeutic protein. Triplicate aliquots from each sample(from Amicon-filtered harvest through drug substance (DS), as well as ablank digest) were diluted to 1 mg/mL in 50 mM Ambic, and mixed 1:1 withRapigest (Waters, reconstituted with 50 mM Ambic to a concentration of0.1%), creating a final solution of 0.5 mg/mL protein in 0.05% Rapigest,50 mM Ambic. Samples were incubated at 60° C. for 15 minutes to ensuredenaturation of the proteins. Reduction of the disulfides was performedwith 10 mM 1,4-dithiothreitol (DTT) (Pierce) in 50 mM Ambic at 60° C.for 1 hour. Samples were allowed to cool to room temperature beforealkylation with 20 mM 2-Iodoacetamide (IAA) (Pierce) in 50 mM Ambic andincubated at room temperature in the dark for 30 minutes. Lys-C(Promega, 15 μg/vial) was reconstituted in 50 mM Ambic at aconcentration of 0.125 μg/μL and added to each sample at anenzyme:substrate ratio of 1:50. The samples were incubated overnight at37° C. The next morning, trypsin (Promega, 100 μg/vial) solution wasprepared at 0.4 μg/μL in 50 mM Ambic and added to the sample at a 1:25ratio and incubated at 37° C. for 3 hours. Rapigest was removed by acidcleavage with the addition of 2.05% formic acid (EMD Millipore). Thesamples were incubated for 30 minutes at 37° C. before being centrifugedat 12,000 rpm for 15 minutes to pellet the cleaved Rapigest and anyundigested protein. Each autosampler vial was prepared with 20 μg ofdigested sample, 400 fmol of Hi3 E. coli standard (Waters) and dilutedto a final volume of 60 μL with 0.1% formic acid for injection on theLC-MS.

The digested samples (30 μL or 10 μg on column) were injected onto aWaters ACQUITY H-Class UPLC system attached to a Waters XEVO G2-XS QTofmass spectrometer for LC-MS analysis using a 1.7 μm CSH C18 Column (2.1mm×150 mm, Waters). The mass spectrometer was set to collect MSE dataover the mass range of 50 to 2000 with 0.3 second scans in sensitivitymode. All samples were run in random order within cleanliness stage(i.e., blanks and drug substance randomized together and run first;mid-process column eluate samples randomized and run next; and cellculture harvest randomized and run last). The UPLC used water andacetonitrile (ACN) with 0.1% formic acid as additives and a gradient of5 to 40% ACN over 30 minutes, ramping to 85% ACN over 5 minutes, holdingfor 2 minutes, and returning to initial conditions over 3 minutes, andholding for 5 minutes with a flow rate of 250 μL/min and total gradienttime of 45 minutes.

The MS data files were imported into Progenesis Qi for Proteomicssoftware (Nonlinear Dynamics) for peak picking and precursor/product ionalignment before being searched against a sequence database using theMSE search algorithm (Waters). The database combined therapeutic proteinproduct sequences, common contaminant proteins, the Chinese hamsterUniprot database, and a reversed version of each (69,916 totalsequences). Identifications were performed with the requirement of 3fragments per peptide, 7 fragments per protein, and at least 2 peptidesper protein with a 4% or less FDR. Further filtering was employed toobtain <1% FDR using a peptide score >5 and peptide mass error <10 ppm.Generally, the peptide identifications were made in the most upstreamprocess samples, e.g., harvest samples, but this was not always thecase. Peptides that did not match the trend for the protein were notincluded for quantitation. The proteins were quantified based on theabundance of the top three peptides per protein (over all threeinjections) compared to the top three peptides for ClpB (Hi-3) or thelinearly-behaving peptides to the product (Mid-3) to determine therelative amount of proteins present in each sample.

Results and Discussion:

One risk of using proteomics discovery software tools for the analysisof relatively low-abundance HCPs in a therapeutic protein productionmixture is that abundant ions from the therapeutic protein can beincorrectly identified as HCPs. Recently it has been shown thatartifacts on abundant proteins can be identified incorrectly as otherproteins at a rate that is much higher than the decoy rate (Kong et al.Nature methods. 2017; 14(5):513-520). It is also possible that HCPs mayalso be identified correctly at the protein level, but some peptides tothose HCPs might be incorrect or could be interfered with by other ions,so they should not be used for quantitation.

The method disclosed herein uses the orthogonal physiochemical propertyof the intensity pattern throughout the purification process to filterout such false positive identifications as well as peptides withinterferences. Within Progenesis Qi for Proteomics, each peptide waslisted along with the identification score and mass accuracy, as well asa visualization of the peptide trend in the entire experiment. An imageshowing the detection of each peptide in m/z and retention time was alsoshown so that other interfering ions can be observed. Peptides having,e.g., observed interference, were not used for quantification.

The LC-MS analysis time for the analysis of samples from the entirepurification process was completed in about two days (LC-MS analysistime for a single sample was about 45 minutes). Manually validating theidentification of all peptides, including peptides from HCPs, tookapproximately two weeks. After validation, a final list of identifiedHCPs from the therapeutic protein production samples was quantified. Asshown in FIG. 4, common HCPs were identified between seven productionlots. Statistical analyses were performed to understand the purificationprocess, e.g., Principal Components Analysis (PCA), which provided anillustration of the removal of the HCPs during each step of thepurification process. Further analysis of the HCPs included hierarchicalclustering, which groups proteins that behave similarly during thepurification process. The hierarchical clustering allowed forinvestigation of nodes to discover which proteins are being removedduring each step in the purification process. These data were used tounderstand and optimize the purification processes to ensure thatspecific HCPs were eliminated from the final therapeutic proteinproduct. HCPs that were not adequately purified from the therapeuticprotein were removed by optimizing the purification process based on,e.g., the physiochemical properties of the HCPs, including pI, molecularweight, hydrophobicity, activity, and immunogenicity. The software wasalso used to automatically monitor the presence of HCPs over the courseof a purification process.

The biggest source of variability in the absolute quantitation of HCPsbetween assay occasions has been either the amount of spiked internalstandard or the amount of that peptide-level internal standard to theHCPs, which are digested proteins. The spiked internal standard wasusually the Hi-3 ClpB peptides that are meant to be used for absolutequantitation. Great care was taken to solubilize, aliquot, and store thestandard, but variability may have occurred due to pipetting differencesbetween, e.g., operators or changes in enzymatic digestion between assayoccasions. Instead of using a spiked internal standard, as shown herein,peptides of the therapeutic protein can be used, however, the mostabundant peptides from the therapeutic protein are often in an abundancethat is beyond the dynamic range of the mass spectrometer, i.e. the mostabundant peptides of a therapeutic protein may over saturate thedetector of the mass spectrometer. Using the most abundant peptidemethod (Hi-3 method), the most abundant peptides were observed to havenon-linear behavior due to, e.g., detector saturation. In contrast, thepeptides in the middle of the ionization distribution, which are calledthe Mid-3 peptides, were observed to have a linear behavior of signalbased on abundance. As shown herein, the Mid-3 peptide method, whichincorporates data from the Mid-3 peptides, yielded a lower error anddisplayed linear behavior up to 18 μg on-column.

What is claimed is:
 1. A method for absolute quantification of a firstpolypeptide in a sample comprising a plurality of polypeptidescomprising the first polypeptide and a second polypeptide, wherein thefirst polypeptide is at least 10-fold lower in abundance than the secondpolypeptide, the method comprising: (a) analyzing peptide products ofthe plurality of polypeptides at a plurality of sample loadingquantities using a liquid chromatography/mass spectrometry (LC/MS)technique to obtain MS signals of ions of the peptide products of theplurality of polypeptides at each of the plurality of sample loadingquantities, wherein the plurality of sample loading quantities comprisesa first sample loading quantity and a second sample loading quantity,and wherein the first sample loading quantity is greater than the secondsample loading quantity; (b) calculating the average or sum of an MSsignal for: (i) a top set of n number of qualified ions of peptideproducts of the first polypeptide with the highest MS signals at thefirst sample loading quantity (A); (ii) a top set of n number ofqualified ions of peptide products of the second polypeptide with thehighest MS signals at the second sample loading quantity (B); (iii) amiddle set of m number of qualified ions of peptide products of thesecond polypeptide at the first sample loading quantity (C); and (iv)the middle set of qualified ions of peptide products of the secondpolypeptide at the second sample loading quantity (D), wherein A, B, C,and D are all calculated using the average or all calculated using thesum of the MS signal; and (c) determining an absolute quantity of thefirst polypeptide in the first sample loading quantity based on thefollowing formula:[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)], or mathematical equivalents thereof.
 2. A methodfor absolute quantification of a first polypeptide in a samplecomprising a plurality of polypeptides comprising the first polypeptideand a second polypeptide, wherein the first polypeptide is at least10-fold lower in abundance than the second polypeptide, the methodcomprising: (a) obtaining MS signals of ions of peptide products of theplurality of polypeptides, wherein said MS signals of ions of thepeptide products are obtained by analyzing the peptide products of theplurality of polypeptides using a liquid chromatography/massspectrometry (LC/MS) technique, wherein MS signals of the peptideproducts are obtained for each of a plurality of sample loadingquantities comprising a first sample loading quantity and a secondsample loading quantity, and wherein the first sample loading quantityis greater than the second sample loading quantity; (b) calculating theaverage or sum of an MS signal for: (i) a top set of n number ofqualified ions of peptide products of the first polypeptide with thehighest MS signals at the first sample loading quantity (A); (ii) a topset of n number of qualified ions of peptide products of the secondpolypeptide with the highest MS signals at the second sample loadingquantity (B); (iii) a middle set of m number of qualified ions ofpeptide products of the second polypeptide at the first sample loadingquantity (C); and (iv) the middle set of qualified ions of peptideproducts of the second polypeptide at the second sample loading quantity(D), wherein A, B, C, and D are all calculated using the average or allcalculated using the sum of the MS signal; and (c) determining anabsolute quantity of the first polypeptide in the first sample loadingquantity based on the following formula:[(A)/(C)]*(mole of the second polypeptide at the first loadingquantity)*[(D)/(B)], or mathematical equivalents thereof.
 3. The methodof claim 1 or 2, wherein the average of the MS signal is used fordetermining the absolute quantity of the first polypeptide.
 4. Themethod of claim 1 or 2, wherein the sum of the MS signal is used fordetermining the absolute quantity of the first polypeptide.
 5. Themethod of any one of claims 1-4, wherein the middle set of qualifiedions of peptide products of the second polypeptide is selected based onquantification error of the qualified ions of peptide products of thesecond polypeptide from the plurality of sample loading quantities, orthe first sample loading quantity and/or the second sample loadingquantity.
 6. The method of any claim 5, further comprising selecting themiddle set of qualified ions of peptide products of the secondpolypeptide.
 7. A method for selecting a set of qualified ions ofpeptide products for absolute quantification of a first polypeptide in asample comprising a plurality of polypeptides comprising the firstpolypeptide and a second polypeptide, wherein the first polypeptide isat least 10-fold lower in abundance than the second polypeptide, themethod comprising: (a) analyzing the peptide products of the pluralityof polypeptides at a plurality of sample loading quantities using aliquid chromatography/mass spectrometry (LC/MS) technique to obtain MSsignals of ions of the peptide products of the plurality of polypeptidesat each of the plurality of sample loading quantities, wherein theplurality of sample loading quantities comprises a first sample loadingquantity and a second sample loading quantity, and wherein the firstsample loading quantity is greater than the second sample loadingquantity; and (b) selecting a middle set of m number of qualified ionsof peptide products of the second polypeptide, wherein the middle set ofqualified ions of peptide products of the second polypeptide is selectedbased on quantification error of the qualified ions of peptide productsof the second polypeptide from the plurality of sample loadingquantities, or the first sample loading quantity and/or the secondsample loading quantity.
 8. The method of claim 7, further comprisingselecting a top set of n number of qualified ions of peptide products ofthe second polypeptide with the highest MS signals at the second sampleloading quantity.
 9. The method of any one of claims 1-6 and 8, whereineach of the top set of qualified ions of peptide products is differentthan each of the middle set of qualified ions.
 10. The method of any oneof claims 1-9, wherein the MS signal is ionization intensity.
 11. Themethod of any one of claims 1-9, wherein the MS signal is peak height.12. The method of any one of claims 1-9, wherein the MS signal is peakarea.
 13. The method of any one of claims 1-9, wherein the MS signal ispeak volume.
 14. The method of any one of claims 1-13, wherein thepeptide products of the plurality of polypeptides in the sample areobtained via sample digestion prior to analyzing the peptide productsusing the LC/MS technique.
 15. The method of any one of claims 1-14,further comprising obtaining the sample.
 16. The method of any one ofclaims 1-15, further comprising processing the plurality of polypeptidesin the sample to produce the peptide products.
 17. The method of claim16, wherein processing the sample comprises one or more of thefollowing: (a) centrifuging the sample to isolate the plurality ofpolypeptides; (b) purifying the plurality of polypeptides in the sample;(c) removing from the sample components incompatible with subsequentprocessing and the mass spectrometry analysis; (d) digesting theplurality of polypeptides to produce the peptide products; and (e)purifying the peptide products.
 18. The method of any one of claims1-17, wherein the LC/MS technique comprises separating the peptideproducts via a liquid chromatography technique.
 19. The method of anyone of claims 1-18, wherein the LC/MS technique comprises processing theobtained MS signals of the peptide products.
 20. The method of any oneof claims 1-19, wherein the LC/MS technique further comprises one ormore the following: (a) identifying the peptide products by amino acidsequence; (b) identifying the first polypeptide by a protein identifier;and (c) identifying one or more of the plurality of polypeptides by aprotein identifier.
 21. The method of any one of claims 1-20, furthercomprising determining the absolute quantity of the second polypeptide.22. The method of any one of claims 1-21, wherein the first polypeptideis a host cell protein.
 23. The method of any one of claims 1-22,wherein the first polypeptide is a biomarker.
 24. The method of any oneof claims 1-23, wherein the first polypeptide is at least 100-fold lowerin abundance than the second polypeptide.
 25. The method of any one ofclaims 1-24, wherein the second polypeptide is a recombinant polypeptideproduced by a host cell.
 26. The method of any one of claims 1-25,wherein the second polypeptide is a therapeutic polypeptide.
 27. Themethod of any one of claims 1-26, wherein the sample is a cell culturesample.
 28. The method of any one of claims 1-26, wherein the sample isa blood or serum sample.
 29. The method of claim 28, wherein the secondpolypeptide is serum albumin.
 30. The method of any one of claims 1-29,wherein the sample is a pharmaceutical product or an intermediatethereof.
 31. The method of any one of claims 1-30, wherein the samplehas been purified or enriched.
 32. The method of any one of claims 1-31,wherein the peptide products are tryptic peptide products of theplurality of polypeptides.
 33. The method of any one of claims 1-32,wherein the plurality of sample loading quantities comprises sampleloading quantities in the range of about 0.1-25 μg total protein. 34.The method of any one of claims 1-33, wherein the first sample loadingquantity is about 10 μg total protein.
 35. The method of any one ofclaims 1-34, wherein the second sample loading quantity is about 1 μgtotal protein.
 36. The method of any one of claims 1-35, furthercomprising selecting the second sample loading quantity based on MSsignals of the second set of peptide products.
 37. The method of any oneof claims 1-36, further comprising selecting the first sample loadingquantity based on MS signals of the first set of peptide products. 38.The method of any one of claims 1-37, wherein each of the plurality ofsample loading quantities has the same total volume.
 39. The method ofany one of claims 1-38, wherein n is 1 or greater.
 40. The method of anyone of claims 1-39, wherein n is
 3. 41. The method of any one of claims1-40, wherein m is 1 or greater.
 42. The method of any one of claims1-41, wherein m is
 3. 43. The method of any one of claims 1-42, whereinthe middle set of qualified ions of peptide products of the secondpolypeptide are selected based on the sequences of each of the peptideproducts.
 44. A method for detecting a contaminate polypeptide in theproduction of a therapeutic polypeptide, the method comprising: (a)obtaining a sample comprising the therapeutic polypeptide; (b)determining if the contaminate polypeptide is present in the sample;wherein the presence of the contaminate polypeptide is based on theabsolute quantification of the contaminate polypeptide in the sampleusing the methods of any one of claims 1-43.